TW201249477A - Methods of making liposomes, liposome compositions made by the methods, and methods of using the same - Google Patents

Abstract

A method of preparing liposomes, liposome compositions formed by the process, and methods of using the liposome composition are provided herein.

Description

201249477 VI. INSTRUCTIONS: Cross-reference to related applications This patent application claims the benefit of the provisional patent application No. 61/430,024 (filed on January 5, 2011). The full text of the case is cited by reference. Into this article. TECHNICAL FIELD OF THE INVENTION The content described herein relates to a method for preparing a liposome comprising an active agent or a desired compound taken up in a liposome, and relating to the use of the liposome composition method. [Prior Art] Liposomes are spherical self-closing small vesicles composed of amphiphilic lipids. Liposomes have been extensively studied and used as carriers for the in vivo administration of pharmaceutical agents, compositions and compounds. The liposomes contain at least one occluded lipid bilayer membrane defining an aqueous compartment. Liposomes have long been used for drug delivery by encapsulating a water soluble drug in an internal aqueous compartment and/or encapsulating a water insoluble agent in a lipid bilayer. The liposome can be a monolayer (uniilamellar) (having a lipid bilayer membrane) or a multilamellar (a bilayer having two or more concentric rows of IJ). Various methods for preparing liposomes and encapsulating therapeutic agents in liposomes are well documented (see, for example, U.S. Patents 3,93 2,65 7 , 4,3 11,712 and 5,0 1 3,5 56 No. All of the above cases are incorporated herein by reference. The known method includes the reverse phase evaporation method described in U.S. Patent No. 4,23,5,8,1,5,,,,,,,,,, Many of the known methods for preparing pharmaceutically acceptable liposomes are not satisfactory for health care liposomes (typically for oral delivery), to some extent due to non-food grade ingredients in medicinal liposome compositions. usage of. Further, many of the known methods use a heating step such as heating to dissolve the lipid, and the heating step increases the temperature of the lipid solution. These methods may affect the performance or efficacy of the agent, compound or composition, particularly agents that are sensitive to heat and/or oxidation. There is therefore a need for additional methods for preparing liposomes for use in healthcare and pharmaceuticals. SUMMARY OF THE INVENTION The aspects described and illustrated below, and the embodiments thereof, are illustrative and illustrative and are not limiting of the scope of the invention. Those skilled in the art will understand other limitations of the relevant art when reading the invention. In one aspect, the invention provides a method of making a liposome having a captured agent. Generally, an appropriate amount of the lipid-forming lipid is dissolved in an aqueous solvent at or about room temperature to form a lipid solution. Alternatively, the captured agent is dissolved in an aqueous solvent at or about room temperature to form a solution containing the agent. The aqueous solvent used to dissolve the lipid and the captured agent may be the same or different. In one embodiment, the aqueous solution contains an alcohol. In another embodiment, the aqueous solution contains ethylenediaminetetraacetic acid (EDTA)®. In one embodiment, the solution containing the agent is filtered. -6- 201249477 In other embodiments, the lipid solution and the solution containing the drug are combined. In other embodiments, a lipid solution is injected into the solution containing the agent with mixing. In other embodiments, the resulting solution is allowed to hydrate for a suitable period of time. In one embodiment, the solution is allowed to hydrate for at least one hour with frequent mixing. In other embodiments, additional lipids and/or thickeners are added to form a gel. In one embodiment, additional lipids and/or thickeners are added after the hydration step. In an exemplary embodiment, the agent is selected from the group consisting of ascorbic acid or a salt or ester thereof, glutathione (GSH), and alpha-dithiooctanoic acid (ALA). In a further aspect, the invention provides a liposome composition for administration of an active agent. In a convenient embodiment, the liposome composition is formed by cold working. In the practice system, the processing is carried out at room temperature, without heating and/or without heating. In the practice system, the active agent is a medicament for health care, dietary supplementation or medicinal use. In a further embodiment, the active agent is selected from the group consisting of ascorbic acid or a salt thereof or vinegar, GSH and ALA. In other embodiments, the liposome of the liposome composition has a selected average particle size of from about 200 to 500 nm. In another embodiment, the liposome of the liposome composition comprises at least about 18 w/w% of the lipid forming the vesicle. In another embodiment, the lipid used to form the sachet is at least about 45 to 50% phospholipid choline. In other embodiments, the phospholipid 201249477 choline is derived from soy or egg. In other embodiments, the phospholipid choline is a high purity phospholipid choline. In a further embodiment, the liposome composition comprises a thickening agent and/or an emulsifier. In the practice system, the thickener and/or emulsifier comprises Sanxian gum and TweenTM 80. In another aspect, the invention provides a unit dosage form comprising a unit dosage package and a liposome composition contained within the package. In another aspect, the present invention provides scurvy, cardiovascular disease, cerebrovascular disease, cancer, age-related macular degeneration, cataract, gout, heavy metal toxicity in a subject treated with ascorbic acid or a salt thereof taken from a liposome. Or the method of diabetes. In one embodiment, a high dose of the liposome composition is administered. In a further aspect, the present invention provides cancer, hepatic insufficiency, malignancy, AIDS, trauma, burns, sepsis, lung disease, Parkinson's disease, diabetes, AIDS, which are treated by GSH in a liposome. Hyperthermia, schizophrenia, swollen fibrosis, heart attack and stroke, seizures, sickle cell anemia, bipolar disorder, chronic fatigue syndrome, autism and immune-related diseases. In one embodiment, a high dose of the liposome composition is administered. In another aspect, the present invention provides an oxidative stress or injury, diabetes, liver disease, inflammation, neurodegenerative disease, such as Alzheimer's disease, cardiovascular disease, surrounding, which is treated or prevented by ALA taken in a liposome. Methods of nerve damage, schizophrenia, obesity, cancer, and hypertension. In the implementation system, high doses of the liposome composition are administered. In addition to the foregoing exemplary aspects and implementation systems, further aspects and implementations will be understood by reference to the following description. [Embodiment] Detailed Description of the Invention I. Definitions In the Invention The terms "active agent", "active compound" and "compound" are used to mean a composition or compound suitable for capture in a liposome. The aforementioned terms include health care, dietary supplementation and medicinal use. The agents contemplated for use in the methods and compositions described herein are quite different and include agents suitable for therapeutic and diagnostic use. "Health food" as used in the present invention refers to food or food that provides health and/or medical benefits. These benefits can be physiological, therapeutic, preventive or diagnostic benefits. Health foods include food, but are not limited to herbal medicines and dietary supplements. As used herein, "dietary supplement" refers to a compound or composition that supplements a diet and provides nutrients. Dietary supplements include, but are not limited to, vitamins, minerals, herbal and other herbal products, fiber, fatty acids. Amino acid or peptide. "Pharmaceutical" or "agent" as used in the present invention refers to any chemical substance or composition intended for medical diagnosis, cure, treatment or prevention of a disease. "Liposome composition" means Is a liposome comprising an agent, compound or composition 201249477 that is at least partially captured in an aqueous space and/or a lipid bilayer. "Forming a lipid for Xiaoxun" refers to a liposome composition capable of forming a stable Any lipid of a part of the object. Such lipids typically include one or two hydrophobic mercapto hydrocarbon chains or steroid groups and may contain a chemical reactive group such as an amine, acid, ester, aldehyde or alcohol on its polar head group. The hydrocarbon chain is typically about 14 to 22 carbon atoms in length and can have different degrees of unsaturation. As used herein, "high dose" and "extra large dose" mean that the dose of the active agent or compound is greater than the officially recommended dose. In the implementation system, '"high doses' and 'extra large doses' refer to recommended doses (eg, government agencies such as US Food and Drug Administration (FDA), Department of Health in the United Kingdom, and the European Union The dose is about 5 to 500 times larger than the agent fi. In other embodiments, "high dose" and "extra large dose" refer to about 10 to 1000 times, 10 to 500 times, 10 to 250 larger than the official recommended dose. Doses, 10 to 100 times, 10 to 50 times (inclusive). "Treatment", "treatment" and the like as used in the present invention generally mean that the symptoms are effectively reduced by administration and/or Or a liposome composition that reduces the severity of the condition to achieve the desired pharmacological and/or physiological effect. This effect may be preventive and/or partial in terms of completely or partially preventing the disease or its symptoms. This effect may be a therapeutic cure for the complete cure of the disease and/or may be attributable to the deleterious effects of the disease. The "treatment" used in the present invention encompasses mammals (especially humans) -10- 201249477 Any treatment for a disease or condition, and includes: preventing the onset of a subject who may be prone to disease but not diagnosed with the disease; inhibiting the disease, ie preventing its development; or alleviating the disease, ie causing the disease to subside. More specifically, ascorbic acid In contrast, 'treatment' can mean therapeutically detectable and beneficial effects in patients with scurvy 'cardiovascular disease, cerebrovascular disease, cancer, age-related macular degeneration, cataracts, gout, heavy metals ( For example, lead and arsenic toxicity and diabetes. "Effective amount", "amount effective" or "therapeutically effective amount" as used in the present invention refers to a medicament that is captured in a liposome. The amount is defined as the amount or dose of the liposome composition which satisfies the therapeutic efficacy, for example, the amount which satisfies the symptoms of preventing, treating or ameliorating the disease or condition. "Pharmaceutically acceptable" as used in the present invention means, for example, a carrier' The diluent or excipient can be compatible with the other ingredients of the formulation and is generally safe or acceptable for administration to the recipient. The drug does not cause an undesired adverse body reaction. "Room temperature" as used in the present invention means a temperature range in the range of from about 10 ° C to 37.7 8 (:, conveniently about 15.5 ° C to 27 °c, more suitably about 18.3. (: to 26.7. (: (including 26.7 ° C). The appropriate "room temperature" range is about 20 ° C to 30 ° C (including 30 ° C). In a specific implementation In the system, "room temperature, can be about 1 5.5 t, 1 8.3 ° C, 1 8.9 ° C, 1 9.4 ° C · 2 0 ° C > 2 0.6 ° C > 2 1. 1 ° C > 2 1 . 7 °C > 2 2.2 °C > 2 2.8 °C ' 2 3.3 °C, 2 3.9 °C, 2 4.4 °C, 2 5〇C > 2 5.6 °C, 2 6.1 °C, 26.1 °C ' 26.7〇C · 27.2 °C « 27.8〇C * 27.8〇C * 28.3〇C 1 28.9〇C > -11 - 201249477 29.4°C ' 30°C , 30.6°C , 31.1°C , 31.7 °C and 32.2 °C. "High temperature" as used in the present invention refers to a temperature above room temperature. In a non-limiting embodiment, "high temperature" refers to about 25. (: to about 60 ° C. In other non-limiting implementations In the system, "high temperature" means from about 25 ° C to about 50 ° C. In a particular non-limiting embodiment, "high temperature" may mean 40 ° C or 50 ° C » as described in the present invention The composition may be administered alone or in a composition with a conventional excipient such as a pharmaceutically or physiologically acceptable organic or inorganic carrier material suitable for oral, enteral or parenteral administration. Not adversely reacting with the compositions used in the methods of the invention. Suitable pharmaceutically acceptable carriers include: water, saline solutions (eg, Ringer's solution), alcohols, oils, gelatins, and carbohydrates, for example Lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and vinylpyrrolidine. Such formulations can be sterilized and, if desired, and do not adversely react with the compositions used in the methods of the present invention. Additives: lubricant, anti-proof Agents, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants and/or perfumes. The formulations may also be combined with other active substances to reduce metabolic degradation, if desired. The "subject" used has its usual meaning and includes primates (such as human and non-human primates), experimental animals (such as chewing animals such as mice and rats), and livestock (for example) Cows, pigs, sheep and horses) and livestock (such as dogs and cats). For liposome compositions containing captured vitamin C, "objects of study" usually refer to animals that cannot synthesize ascorbic acid. Mammalian package for synthesizing ascorbate-12- 201249477 Human genus (including human, monkey, and cockroach) such as primates. "Vitamin C" as used in the present invention refers to L-ascorbic acid and L-ascorbate, and Refers to ascorbic acid inorganic salt or ascorbate. The "GSH" used in the present invention refers to glutathione, (2S)-2-amino-4-{[(lR) -1-[(carboxyl) Methylaminomethane]-2-thioethyl]methylamine guanidine} "ALA" as used in the present invention refers to α-dithiooctanoic acid, two mirror image isomers R-(+)-dithiooctanoic acid (RLA) and S-(-)-dithiooctanoic acid (SLA). A racemic mixture, an effective mirror image isomer of RLA or an inorganic salt of ALA. "ATP" as used in the present invention means adenosine-51-triphosphate, and further refers to an inorganic salt of ATP. "Companion" merely means that two conditions exist and should not be construed as indicating that one must have a causal relationship with the other. II. Method for preparing liposome compositions A typical method for preparing liposomes is included in the method. The lipid or liposome composition is heated during at least one step (typically two steps). First, the lipid solvent composition is typically heated to a high temperature to increase the solubility of the lipid in the solvent. Lipids used in the manufacture of liposome compositions are typically supplied as granules or slabs of waxy materials, and heating solvents are generally required to dissolve the lipids. Second, conventional liposome preparation methods require encapsulation of the active agent and/or fractionation of the liposomes, homogenization, ultrasonic treatment or microfluidization homogenization steps. During homogenization (or ultrasonication or microfluidization homogenization, as appropriate, from 13 to 201249477), forcing the lipid-active agent composition to pass through the interaction chamber of the homogenizer at high speed causes heat to cause the lipid-active agent mixture to be heated to high temperature. Although the composition cools to a lower temperature once it leaves the interaction chamber, most of the composition remains hot after a period of homogenization. Many active agents, lipids and compounds, including vitamins and other nutritional supplements (like vitamin C, glutathione and ALA) are sensitive to oxidation (accelerating at high temperatures). For example, vitamin C is sensitive to heat, light, and oxygen, and thus rapidly degrades during homogenization during liposome application and high temperatures caused by heating. It will be appreciated that the use of heat to prepare a medicament that is captured in a liposome will allow most of the agent to degrade during processing. In one embodiment, the cold working method solves this problem by providing a single step at or near room temperature (about 20 to 25 ° C) and/or forming small size liposomes without heating. In a convenient embodiment, "cold processing" refers to a method of forming a liposome without a heating and/or heating step. In another embodiment, "cold processing" refers to a method of forming liposomes at or near room temperature. In this embodiment, "cold processing" is synonymous with "room temperature." In other embodiments, The method comprises preparing a liposome without heating. In another embodiment, the method comprises preparing a liposome without a heating step (e.g., one or more of heating a solvent). This step of processing does not include or require any further Processing steps (eg, homogenization or ultrasonic treatment or microfluidization homogenization) to encapsulate the active agent a within the liposome splicing or to fractionate the liposome. Thus, in accordance with conventional methods for forming liposome compositions The agent in the liposome composition prepared by the cold working described in the present invention has significantly less degradation and/or loss of activity when compared to the drug taken in the liposome-14-201249477. In cold working, the lipid is conveniently completely or mostly dissolved in an aqueous solvent or a water-miscible solvent (for example, most alcohols) in the liposome group. The product is used orally, and the aqueous solvent is conveniently a solvent suitable for ingestion. In a system in which the solvent in cold working is an alcohol, the final alcohol content in the final product is preferred to be less than about 10 to 12 wt%. % is calculated based on the weight of the final product. Although alcohol is not necessary to form a stable liposome product, it appears to be helpful in the manufacture of translucent gels. The alcohol further has the benefit of being an effective preservative. Use may also be helpful or effective in forming small liposomes ranging in size from about 200 to 500 nm. Other aqueous solvents are suitable and well known to those skilled in the art. However, some solvents may result in compositions that are made with alcohol. A more turbid appearance. In the subject cold working, the lipid can be dissolved in the alcohol, in some embodiments at elevated temperatures (above room temperature) to accelerate the dissolution of the lipid in the alcohol. However, if the heat dissolves the lipid, then Cooling the lipid solution prior to hydration with the aqueous solution of the active agent (eg, by normal means or using a heat exchanger for large scale processing) To a lower temperature (e.g., room temperature). This processing method is different from the "typical" liposome manufacturing method in which the lipid and the active agent (solution) are heated, for example, as described above. An aqueous solution containing the active agent is prepared in the system at room temperature. Other compounds such as ethylenediaminetetraacetic acid (EDTA) may be included as needed to improve solution characteristics or to improve the solubility of the active agent in water. Water can be purified by any suitable means including, but not limited to, reverse osmosis, deionization, and filtration. Once dissolved, the solution containing the active agent can be further filtered to remove contaminants. A suitable filter is a 0.2 μη film, for example 0.2 μηι Mini Capsule Sterile Filter» available from Pall Life Sciences It should be understood that the pore size of the membrane can be adjusted according to the size of the contaminant to be removed from the solution. The choice of a suitable aperture size filter is a good skill for those skilled in the art. Appropriate filter aperture sizes include, but are not limited to, 0.5 μηη, 0.4 μηη, 0.45 μηι, 0.3 μηι, 0.2 μηι, 0.15 μιη, and 0.1 μηι. Next, in cold working, the lipid solution is added to the aqueous solution of the active agent by injecting or injecting the lipid solution into the aqueous solution of the active agent, optionally under agitation. Stirring can be provided by any suitable means including mechanical agitation (e.g., using a blender) and manual mixing or agitation. After the lipid solution is added to the aqueous solution of the active agent, the lipid hydration should be allowed to continue for at least about half an hour or one hour with frequent agitation. At the end of processing, the resulting liposome composition is conveniently a smooth fluid.

For the purpose of making the liposome composition savory and easy to handle, the liposome nutritional supplement or composition is formulated to have a gel consistency in one embodiment. Additional lipids are added to the composition in one embodiment to increase the viscosity of the liposome composition to a gel-like viscosity. In another embodiment, a thickening agent and/or emulsifier is added to the composition like a gel with or without the addition of more lipid. Suitable thickeners and/or emulsifiers include Sanxian gum, locust bean gum, TweenTM 80 (polysorbate 80), TweenTM-16-201249477 20 (polysorbate 20), TweenTM 40 (polysorbate 40) ),

TweenTM 60 (polysorbate 60), lecithin, emulsifying wax, cetyl alcohol and ceteareth 20 . Further suitable thickeners are described at the website en.wikipedia.org/wiki/Category: Edible_thickening_agents. The use of thickeners and/or emulsifiers can be used to reduce the amount of lipid required to make the gel. Alternatively, in the absence of a thickening agent and/or emulsifier, the amount of lipid can be increased by up to 30 to 35% from the amount necessary to form the liposome to produce a liposome composition having a gel-like consistency. The gel composition is prepared using an excess of lipid to provide a higher active agent encapsulation efficiency. A combination of additional lipids and thickeners and/or emulsifiers can be used to make the gel composition. The addition of excess lipids, thickeners and/or emulsifiers may also have the benefit of making the gel for oral use more palatable. Such compositions may have a honey-like color rather than an orange or brown color of the liposome composition made by other methods. The composition formed by the method of the present invention is also preferably partially or wholly transparent. Table 1 provides exemplary liposome formulations for sodium ascorbate as an active agent. It should be understood that the formulations in Table 1 are for illustrative purposes only. This formulation is non-limiting and may include other ingredients and different ranges. It should be understood that the percentage of active agent may vary depending on the nature of the active agent. -17- 201249477 Table 1: Demonstrated liposome formula w/w% Range of change (wt%) Lipid 1 18.73 10-30 Sodium ascorbate (vitamin C) 22.00 5-25 Water 47.5 qs to 100 Alcohol (200 proof) 11.00 0- 15 Sanxianjiao 0.42 0-2 TweenTM80 0.26 0-2 EDTA 0.005 0.005-0.1 At least 45 to 50% is conveniently a PC, such as Phospholipon 50IP, a non-GMO PC, or Alcolec PC 50. In the HI-liposome composition, the agent, composition or compound is captured in the liposome. The active agent, compound or composition can be encapsulated in a wide variety of liposome compositions. The knowledge of those skilled in the art is sufficient to select the composition and composition ratio of the liposome composition, depending on, for example, the form of the agent (e.g., ascorbic acid, ascorbate, inorganic salts, esters, etc. for vitamin C) and the method of administration. The lipid used to form the liposome composition includes a lipid for forming a small capsule having two hydrocarbon chains (typically a thiol chain) and a polar head group. Such lipids include phospholipids such as phospholipid choline (PC), phospholipid oxime ethanolamine (PE), phosphatidic acid (PA), phospholipid creatinine (PI), and sphingomyelin (SM), wherein the two hydrocarbon chains Typically it is between about 14 and 22 carbon atoms in length and has different degrees of unsaturation. In a convenient embodiment, the lipid is a relatively unsaturated phospholipid (having a -18-201249477, two or three double bonds in the hydrocarbon chain). In a particularly convenient embodiment, the lipid is phospholipid choline. Phosphocholine choline is a phospholipid that incorporates choline as a head group and binds one glycerophosphoric acid and two fatty acids. Phospholipid choline is a major component of biofilms and may be extracted from available sources (such as egg yolk or soy) by known methods. In one embodiment, the lipid is phospholipid choline derived from soybean. In another embodiment, the lipid is phospholipid choline derived from the egg yolk. Suitable phospholipid choline lipids include, but are not limited to, Phospholipon 50IP and Alcolec PC 50 available from the American Lecithin Company (Oxford, CT). It will be appreciated that more than one lipid can be used to prepare the liposome composition. The lipid and ratio options can be varied to achieve the desired degree of fluidity or rigidity to control stability and/or to control the rate of release of the captured agent. In the case where more than one lipid is used to prepare the liposome composition, an appropriate amount of a relatively unsaturated lipid (e.g., PC) should be used to form a stable liposome. In one embodiment, at least 45 to 50 mole percent of the lipid used in the formulation is phospholipid choline (PC). Liposomes may also include lipids derivatized with a hydrophilic polymer such as polyethylene glycol PEG. Stable hydrophilic polymers include: polyvinylpyrrolidone, polyvinyl methyl ether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide , polymethacrylamide, polydimethyl methacrylate, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, poly Indolinamide and hydrophilic peptide sequences. A method of preparing a lipid derivatized with a hydrophilic polymer is known (see, for example, U.S. Patent No. 5,395,619, incorporated herein by reference) The liposomes can be further administered in the form of gelatin, such as gelatin capsules or soft gelatin gels known in the art. The liposome compositions of the present invention described in Examples 4 to 5 and 9 are highly stable. It has further been demonstrated that the liposome compositions of the present invention are highly stable when stored at elevated temperatures (temperature above room temperature). As used herein, "stability" refers to the formulation retaining most of the captured medicament during storage and after storage. In a non-limiting embodiment, the liposomes remain stable after storage for at least about six months or one year. In other non-limiting implementation systems, the liposomes remain stable after storage for at least about one to twelve months (including twelve months). In other non-limiting implementation systems, at least about 1, 2, 3, 4, 5, 6, or 9 months or more. In the practice system, at least or more than about 65 to 100% (including 100%) of the captured agent remains in the liposome after storage. Conveniently, the liposomes retain at least or more than about 65%, 70%, 80% '90% > 95% '9 7%, 98%, 990/〇 or 100% of the captured body after storage. Pharmacy. In the case where the liposomes are stored at elevated temperatures (above room temperature and, for example, 25 to 60 ° C, conveniently about 50 ° C), it is convenient to retain at least about 65% of the captured agent in the liposomes after storage. In one embodiment, the liposomes of the invention have a particle size of from 200 to 600 nm, conveniently from about 200 to 500 nm or from about 300 to 500 nm. See Example 3, cold-formed liposomes have substantially smaller particle sizes than commercially available oral liposomes. Liposomes with smaller particle sizes for oral administration are generally more delicious. Thus, liposomes -20-201249477 formed by this method are more delicious than commercially available oral liposomes. In one embodiment, the liposomes formed by the present method have an average particle size distribution that is 25 to 75% smaller than liposomes formed by other methods (e.g., commercially available liposomes). In a non-limiting embodiment, the liposomes of the invention have 255%, 35 %, 40%, 45% less liposomes (e.g., commercially available liposomes) formed by other methods. , 60%, 65 % or 7 5 % of the average particle size distribution. IV. The captured agent is intended to be used in the method and the liposome composition of the agent, compound and composition are greatly different and include health care agents, including dietary supplements and agents A. Health care agents 1. Ascorbic acid and its salt ascorbic acid It is a water-soluble sugar acid with antioxidant properties. Ascorbic acid and its sodium, potassium and calcium salts are commonly used as food additives due to the nature of the antioxidant. Similarly, fat-soluble ascorbyl esters having long-chain fatty acids (e.g., ascorbyl palmitate or ascorbyl stearate) are commonly used to prevent oxidation of fat. The L-mirror isomer of ascorbic acid (referred to as vitamin C or L-ascorbic acid, the salt of which is L-ascorbate) is an essential nutrient for people who are unable to manufacture ascorbic acid and some other mammals. The lack of vitamin C leads to scurvy in humans. Vitamin C is also required for the synthesis of collagen, L-carnitine and certain neurotransmitters (eg, norepinephrine) -21 - 201249477. Vitamin C is further involved in protein metabolism and is a highly potent antioxidant. Vitamin C has long served as a nutritional supplement because many people do not get enough vitamin C from their diet. The National Health and Nutrition Examination Survey data from 1 999 to 2000 showed that nearly 35% of adults consumed a multivitamin supplement (typically containing vitamin C). This data also indicates that 12% of adults consume different vitamin C supplements (Radimer, et al., Am. J. Epidemiol., (2004), 1 60: 339-49). Vitamin C is the most commonly used dietary supplement. Vitamin C can be used orally in tablets and administered intravenously. Oval medicinal tablets, lozenges, gums, synthetic beverage packs, synthetic vitamin formulations, comprehensive antioxidant formulations, and crystalline powders can be applied to the oral cavity. However, oral administration is generally limited to about 2 to 3 grams per day (g) due to intestinal irritation and diarrhea with larger doses. It has been found that ingestion of vitamin C at doses greater than the level of enteric tolerance may cause diarrhea (Cathcart, Medical Hypothesis (1981), 7:1359-1376) « The level of intestinal tolerance dose of vitamin C varies from individual to individual and can It is in the range of 5 mg (mg) to several thousand mg per 曰. The use of vitamin C, including high or extra doses, has long been advocated for the treatment of a wide variety of diseases and conditions, including cardiovascular disease, cancer, cataracts, gout and the toxicity of heavy metals such as lead and arsenic. It is customary for high doses or extra large doses of vitamin C to be administered intravenously to avoid oral side effects and to increase blood levels to levels above the blood levels typically achieved orally. It is generally believed that the intestinal absorption of vitamin C is regulated by at least one active transporter that is more dependent on the -22-201249477 degree dependence, thereby limiting the amount of vitamin C absorbed (Jacob et al., Nutr. Clin. Care, (2 002) , 5:66-74). Pharmacokinetic simulations predict that a high dose of 3 g of ascorbic acid per 4 hours will produce a peak blood concentration of only 220 micromoles per liter (Padayatty, et al., Ann. Intern. Med. (2004) 140:533- 7). For oral administration, vitamin C has been encapsulated in liposomes to increase absorption and reduce oral side effects. Lipoflow provides liposome vitamin C for conditions of lipid metabolism and atherosclerosis. Each tablespoon of the supplement contains 1 mg of vitamin C encapsulated in the essential phospholipids from soy lecithin. LivOn Laboratories provides a supplement containing 1000 mg of liposomes encapsulated with vitamin C in 100 Orng essential phospholipids. Existing liposome vitamin C formulations contain large particle size liposomes (about 800 to 1 000 tim) that are less orally tasty. Due to the size of the lipid body, the existing formulation is turbid. There is therefore a need for a more delicious and stable oral liposome formulation. In one aspect, the liposome composition is provided with a nutritional supplement or composition comprising L-ascorbic acid, L-ascorbate, ascorbic acid inorganic salt or ascorbyl ester. In one embodiment, the nutritional supplement or composition contains an alkali metal salt of L-ascorbate. Suitable inorganic salts include, but are not limited to, sodium ascorbate, calcium ascorbate, potassium ascorbate, magnesium ascorbate, zinc ascorbate, molybdenum ascorbate, chromium ascorbate, and manganese ascorbate. Due to the buffering of inorganic salts, it may be less irritating than ascorbic acid when ingested. It should be understood that a combination of ascorbic acid inorganic salts can be used. It may be particularly important to administer a high dose or overdose to maintain a recommended intake of cations for inorganic salts at or -23-201249477 close to the recommended foraging guidelines. In another embodiment, the nutritional supplement or composition comprises esterifying with a saturated fat to produce a fat soluble ascorbic acid. One suitable fatty acid is palmitic acid to form ascorbyl palmitate. The L-ascorbic acid, its inorganic salt or ester is conveniently taken up in the liposome. a. High dose In another aspect, the present invention provides a high dose or extra large dose of a liposome composition comprising a nutritional supplement or composition comprising L-ascorbic acid, an ascorbate inorganic salt or ascorbic acid vinegar. The Reference Intake developed by the Institute of Medicine's Food and Nutrition Board for Vitamin C is in the range of 45 to 120 mg per day for adolescents and adults. Smokers are advised to consume an additional 35 mg daily. In 2009, FDA updated the Recommended Daily Intake (RDI) to 60 mg/day (www fda.gov). In general, "high dose" and "extra large dose" refer to a dose of about 1 〇〇 to 1 〇, 〇0 〇 mg/day. "High dose" and "extra large dose" may also be "enteral tolerance doses", which are doses below the dose at which the subject is experiencing diarrhea (see Cathcart, Medical Hypotheses, 7:1 3 5 9- 1 3 76,1981, an exemplary method for determining the "enteral tolerance dose", which is incorporated herein by reference.) In a convenient implementation system, "extra large dose" refers to the dose of ascorbic acid or its salt equal to or A dose greater than 2000 mg/day. In another embodiment, the high dose of the vitamin C-containing liposome composition, including the dose of vitamin C, is recommended for the following recommended intake: National Academy of -24- 201249477

Sciences (90 mg/day in 2000), the Food and Nutrition Board (75 to 120 mg/day), the USDA or other government management unit. In the implementation system, about 100 to 1 〇, 〇〇〇 mg / 曰 dose is administered. In other implementation systems, the dosage is approximately 200 to 2.000 mg/day '200 to 5,000 mg/day '200 to 1 0,000 mg/day, 400 to 10,000 mg/day '400 to 8,000 mg/day, 400 to 6,000 Mg/day, 400 to 5, 〇〇〇mg/day, 400 to 4,000 mg/day, 400 to 3.000 mg/day, 400 to 2,000 mg/day, 500 to 10,000 mg/day, 50,000 to 8,000 mg/ B, 500 to 6,000 mg/B, 500 to 5,000 mg/day, 500 to 4, 〇〇〇mg/day, 50,000 to 3,000 mg/day '500 to 2.000 mg/day '500 to 1, 〇〇〇 Mg/day, 1000 to 10,000 mg/day, 1,000 to 8,000 mg/day '1 to 6,000 mg/day, 1000 to 5.000 mg/day, 1 to 4,000 mg/day, 1 000 to 2,000 Mg/day, 2000 to 10,000 mg/day, 2000 to 6,000 mg/day, 2000 to 5.000 mg/day, 2000 to 4,000 mg/day, 4000 to 10,000 mg/day, 4000 to 8,000 mg/B, 4000 to 6,000 mg / 臼, 4000 to 5.000 mg / day or 5,000 to 1 〇, 〇〇〇 mg / day (including 1 0,000 mg / 曰). For humans this dose should be below LD50. Although oral LD50 is lower in mice (3 3 67 mg/kg), it is 11,900 mg/kg for 'rats (MSDS Data Sheet for ascorbic acid) » It should be understood that this dose can be daily Do it once or several times. Conveniently, more than one dose per day above 1,000 mg is administered at least 30 minutes apart. Although some studies suggest that oral vitamin C concentrations reach a serum saturation of approximately 200 to 240 mg, these studies are usually at 12 or -25- 201249477

The measurement of blood concentration after 2 hours is based on the measurement. Since vitamin C has a half-life of 30 minutes, the true serum saturation should be higher to make high-dose or extra-dose vitamin C feasible. 1991 USD A

A study conducted by the Human Nutrition Research Center found that the concentration of vitamin C in the eyes of the elderly who consumed 200 mg of vitamin C per day was higher than that of the elderly who consumed 148 mg of vitamin C per day, indicating that higher doses were absorbed. And distributed within the organization (Taylor et al., Current Eye Research, 1991, 10 (8): 751-759). Approximately 70 to 90% of oral vitamin C is absorbed at a dose of 30 to 180 mg per day (Vitamin C Fact Sheet at ods.od.nih.gov). However, absorption decreases as the dose increases. Maintain high vitamin C concentrations in cells and tissues, and the highest concentrations found in white blood cells, eyes, adrenal glands, pituitary gland and brain ( Vitamin C Fact Sheet at ods.od.nih.gov) ° Because vitamin C is water soluble (33 g / 100 mL) so it is not stored in the body, even in high doses or extra large doses of vitamin C toxicity is very rare. Vitamin C at high doses may cause dyspepsia (especially on fasting) and diarrhea (news-medical.net/Vitamin-C-Side-Effects.aspx). In the 1986 trial, up to 6 g of ascorbic acid was administered to '29 infants, 93 preschool and school age children, and 20 adults for more than 1400 days. At higher doses, toxicity was observed in 5 adults and 4 infants. Symptoms and symptoms in adults are nausea, vomiting, diarrhea, blushing, headache, fatigue and poor sleep. The main toxic reaction in infants is rash (Widenbauer, Klin. Wschr, 3 3:1 1 5 7,1 936 ). Another study found that consumption of up to ι 每日 daily, 〇〇〇-26- 201249477 mg dose of vitamin C for up to three years is safe (Bendich, et al., J. Am. College Nutr., 1 9 9 5 , 1 4 ( 2 ) : 1 24- 1 36 ). b. Liposomal Compositions Nutritional supplements or compositions can be encapsulated in a wide variety of liposome compositions. The knowledge of the artist is sufficient to select the composition and proportion of the liposome composition, depending on the form of vitamin C (ascorbic acid, ascorbate, ascorbate inorganic salt, ascorbate, etc.) and the method of administration. In one embodiment, the agent encapsulated in the liposome is L-ascorbic acid, an inorganic salt or ester thereof. The liposome compositions of the invention have proven to be highly stable. The liposome compositions of the present invention are further shown to be highly stable when stored at elevated temperatures. Since vitamin C is sensitive to heat (accelerating the oxidation of molecules), it is particularly advantageous to be highly stable when stored at elevated temperatures. "Stabilization" as used in the present invention refers to the retention of most of the vitamin C in the formulation. Conveniently, at least or more than about 65%, 70%, 80% '90% '95% ' 9 7%, 98% '9 9 0/〇 or 100% of the vitamin C ° is retained in the liposome. Where the liposomes are stored at elevated temperatures (e.g., at about 50 °C), it is convenient to retain at least about 65% of the vitamin C in the liposomes. An exemplary liposome composition was prepared by encapsulating sodium ascorbate (18.4 wt%) in a liposome containing Alcolec PC 50 lipid (see Examples 1 and 4). The liposome composition was described in Example 4. Store in a 2 mL vial sealed in N2T and store at room temperature (25. (:) or high temperature (5 ° ° C). At 4, 5, 7 (measuring two vials), 8 and 38 days The weight percentage of ascorbic acid was measured by HPLC from -27 to 201249477. It is seen in Figure 3 that the weight percentage of ascorbic acid in the vial stored at room temperature is highly stable throughout the period. On the 38th, it exists on the 4th day. 99.8% of ascorbic acid is still present in the sample, showing little or no degradation of vitamin C after storage at 25 ° C for 38 days. Conveniently, at least about 1 week, 2 weeks, 3 weeks, 1 month, 2 The liposome retains most of the vitamin C during months, 3 months, 6 months or 1 year. Even at high temperatures, the vitamin C content in the liposome composition is highly stable. See in Figure 3 After storage on the 20th day of 501, about 96% of the vitamin C content at the beginning is still safe. Even after 42 days, 80.8 wt% of ascorbic acid remained in the liposome composition. Furthermore, in Figure 6, it was observed that even at room temperature (25 °C) and high temperature (40 °C) 120 The weight percentage of sodium ascorbate that is captured in the future is stable. This stability is at least comparable to the currently available vitamin C composition. As described in Example 5, the preparation of the non-GMO lipid described in Example 1 was prepared. The storage stability of the liposome composition of the present invention was compared with the vitamin C morphine formula (Lyp〇-SphericTM Vitamin C liposomes) available from LivOn Laboratories. The liposome composition of the present invention and Lypo-SphericTM Vitamin C Liposomes were stored in a 2 mL vial sealed under N2 and stored at elevated temperature (50 ° C). The stability of Lypo-SphericTM Vitamin C liposomes was also monitored as comparisons in the original packaging. At 0, 4, 13 And the weight percentage of ascorbic acid was measured on the 19th. It is seen in Fig. 4 that about 92% of the vitamin C content at the beginning after the fourth day is still stable, and the vitamin C content at the beginning of the 13th day after the start of -28-201249477 Approximately 97% remained stable and approximately 89% of the liposomes remained stable after 19 days. This is comparable to Lypo-SphericTM Vitamin C liposomes, which retained approximately 99-90% in liposomes after 19 days. Vitamin C. As seen in Figure 4, the stability of Lypo-SphericTM Vitamin C liposomes was greater when the liposomes were stored in the original packaging (in the N2T seal), leaving 99.5 wt% ascorbic acid after 19 days. Since the liposome composition of the present invention is comparable in stability to Lyp〇-SPhericTM Vitamin C liposomes, storage of the liposomes of the present invention in a package sealed under the same N2 purge may result in similar stability. Figure 5 is a graph comparing liposome stability of different batches of liposomes of the invention and Lypo·SphericTM Vitamin C liposomes after 90 days. It is seen from Fig. 5 that about 65% of the ascorbate at the beginning of three months at 50 ° C is still effective. This is a higher stability compared to Lypo-SphericTM Vitamin C liposomes. It is seen from Figure 5 that the ascorbate in Lypo-SphericTM Vitamin C liposomes tends to be more downward than the liposome of the present invention. c. Methods of Use In another aspect, the invention encompasses methods of using liposome compositions prepared in accordance with the methods described herein. Of course, the method of use or method of treatment depends on the compound being captured. Several embodiments of the method of use are set forth below, however, the skilled artisan will appreciate that the embodiment is merely exemplary. In one aspect, the present invention provides for treating or preventing scurvy, cardiovascular disease, cerebrovascular disease, cancer, age-related macular portion by administering a dose of vitamin C captured in the plastid of lipid-29-201249477 Pathology, cataracts, gout, toxicity of heavy metals (eg lead, arsenic, cadmium, copper and mercury) and diabetes. In one embodiment, the dose is a high or very large dose of vitamin C taken from the liposome. Vitamin C is an antioxidant and a cofactor in at least eight enzyme reactions. Vitamin C is also required for the synthesis of collagen, an important component of blood vessels, ligaments and bone. Vitamin C also plays a role in the synthesis of norepinephrine, a neurotransmitter. Vitamin C is further involved in the conversion of cholesterol into bile acid, which may imply blood cholesterol levels (Simon et al., Arch. Intern. Med., 2000, 1 60 (7): 93 1 -936). Several individuals and institutions have advocated the use of large doses of vitamin C (Linus Pauling Institute at lpi.oregonstate.edu/infocenter/vitamins/vitaminC/index.ht ml) for the treatment of a wide variety of conditions and diseases. In addition to the treatment and prevention of scurvy, the study pointed out that vitamin C is harmful to cardiovascular disease, cerebrovascular disease, cancer, age-related macular degeneration, cataract, gout, heavy metals (including Gi, Jana (Mahajan et al., Asian) J. of Microbiology, Biotech. & Env. Sci., 2001, 3 ( 1 -2 ) : 95- 1 00 ), Li ( Mahajan et a 1.), Shop (Borane, et a 1., J. Aquatic Biology, 2006, 21 (2): 244-248) and arsenic (Karasawas, et al., Blood, 2005, 105 (1 0): 4004-40 1 2 )) and the treatment or prevention of diabetes is effective. Cardiovascular Disease -30- 201249477 Cardiovascular disease is a disease involving the heart or blood vessels. Cardiovascular diseases include, but are not limited to, hypertension, coronary heart disease (including myocardial infarction and angina), stroke, and heart failure. Coronary heart disease results from atherosclerosis, which is caused by the accumulation of fatty substances such as cholesterol. Coronary heart disease is the single leading cause of death in the United States (American heart.org) Vitamin C has been shown to reduce adhesion of mononuclear leukocytes to the endothelial layer, improve endothelial-dependent nitric oxide production and vasodilation, and reduce vascular smooth muscle cell death ( Honarbakhsh, et al., Br. J. Nutr., 2008, 1-1 9 ). Randomized, double-dose, placebo-controlled studies have demonstrated that subjects treated with vitamin C for coronary heart disease and those with angina, congestive heart failure, high cholesterol, and high blood pressure Vasodilation improvement (Gokce, Circulation, 1999, 99 (25): 3234-3 2 4 0; Versari, et al., Br. J. Pharmacol., 2009, 157 (4): 5 27-536; and Frikke -Schmidt et al., Basic Clin.

Pharmacol. Toxicol., 2009, 1 04 (6): 4 1 9-43 3 ). Several studies have demonstrated the benefits of vitamin C supplementation for the treatment of cardiovascular disease. Plasma vitamin C concentrations related to stroke were monitored in prospective studies of 20,649 men and women with prevalence of stroke between the ages of 40 and 79. The subjects in the top quartile of baseline plasma vitamin C concentrations had a 42% lower risk of stroke than the subjects in the bottom quartile (Myint, et al., Am. J. Cling. Nutr. , 200 8, 87 ( 1 ) : 64-69 ). Vitamin C (120 mg) plus molybdenum was provided daily for 5 to 6 years in the Linxian trial, and the risk of cerebrovascular death 201249477 was reduced by 8 °/ό during the 10-year period following the cessation of supplementation (Qiao, et al., J. Natl. Cancer Inst., 2009, 101 (7): 507-518). A pool of nine studies found that people who consumed 2700 mg/day of vitamin C had a 25% lower risk of coronary heart disease (Knekt, et al., Am. J. Clin. Nutr., 2004, 80 ( 6 ) :1 508-1 520 ). Cancer epidemiological evidence suggests that higher fruit and vegetable consumption is associated with a lower risk of most types of cancer, perhaps partly because of its high vitamin C content (Li, et al., J. Nutr., 2007, 137: 2171-84). Most case-control studies have found an inverse association between dietary vitamin C intake and lung cancer, breast cancer, colon or rectal cancer, stomach cancer, oral cancer, laryngeal or throat cancer, and esophageal cancer (Jacob, et al., Nutr. Clin. Care, 2002, 5: 66-74). The plasma concentration of vitamin C in people with cancer is also lower than in the control group (Carr, et al. Am. J. Clin. Nutr., 1 999, 69:1 086-1 07). Intravenous administration of vitamin C has been shown to produce a plasma concentration of 2,600 micromoles per liter (Padayatty, et al., CMAJ 2006, 1 74: 937-942). Such high concentrations are selective cytotoxic to isolated cancer cells (Li, et al., J Nutr 2007, 1 3 7:2 1 7 1 -84;

Shekelle, et a 1., Evidence Report/Technology Assessment No. 83 AHRQ Publication No. 03-E043. Rockville, MD: Agency for Healthcare Research and Quality, 2003) ° The study of mice suggests the pharmacology of intravenous vitamin C Dosage is promising in the treatment of refractory cancers by different methods (Chen, et al., Proc Natl -32- 201249477)

Acad Sci USA 2008,1 05:1 1 1 05- 1 1 1 09 ). High concentrations of vitamin C can act as oxidizing agents and produce hydrogen peroxide that is selectively toxic to cancer cells (Chen et al, 2009; Chen, et al., Proc Natl Acad Sci USA, 2005, 102: 13604-13609; And Chen et al., Proc Natl Acad Sci USA, 2007, 1 04: 8749-8754). There are also very few cases of end-stage cancer patients with very long survival times after high-dose intravenous vitamin C administration (Levine, et al., Free Radic, Biol. Med., 2009, 47:27-29) . Diabetes Diabetes is a group of diseases characterized by high blood sugar concentrations caused by insufficient ability to make and/or use insulin in the body. In the United States alone, 23.6 million people have diabetes (diabetes.org) that includes type 1 diabetes, type 2 diabetes, and gestational diabetes. Type I diabetes is usually diagnosed in children and young people and is characterized by the inability to produce insulin. The treatment of type 1 diabetes usually requires insulin therapy. A study of type 1 diabetes patients in 993 showed that intravenous vitamin C administration produced a bimodal plasma concentration curve of insulin (Kodama, et. al., In Vivo, 1993, 7(6A): 535-542). These results indicate that vitamin C may contribute to the treatment of diabetes by stimulating the insulin mechanism. In addition, vitamin C can be used to prevent or reduce vascular damage caused by diabetes in patients with poor glycemic control. Type II diabetes is the most common form of diabetes. The type of diabetes is characterized by the inability of the body to make enough insulin or the inability to use insulin effectively. -33- 201249477 . Yazd Diabetes Research Center's study of type 2 diabetic patients receiving 1000 mg of vitamin C supplement daily showed fasting blood glucose, triglycerides, and 42 days per patient receiving 500 mg of vitamin C. Low-density lipoprotein, glycated hemoglobin, and serum insulin concentrations were significantly reduced (eight € let 1^111 b eight 1 (161^1^, 61 & 1_, 111〇11 & 1111.?46 (1· Res·, 2007, 1 26 ( 5 ) : 4 7 1 -474 ). The mean fasting blood glucose concentration decreased from 169.33 mg/dl to 144.80 mg/dl. The average HbAiC decreased from 8.82% to 7.66%. The average LDL decreased from 1 3 0.95 mg/dl to 125.9 1 mg/dl. The insulin concentration was reduced from 16.91 microunits/mL to 8.77 microunits/mL. These results show that consumption of at least about 1,000 mg of vitamin C supplement per day is beneficial for reducing blood glucose and lipids in type 2 diabetic patients. Age-related macular degeneration and cataract age-related macular degeneration (AMD) and cataract are two of the main causes of vision loss in the elderly. Age-related macular degeneration is a disease that affects the central retina and leads to a gradual loss of central vision in agility. Back Exudative senile macular degeneration occurs when abnormal blood vessels grow under the middle region of the retina and cause damage to the central region of the retina. Atrophic senile macular degeneration occurs when the photoreceptor cells in the retina are slowly damaged and the central vision is blurred. Cataract is a turbidity developed in the lens of the eye or its capsule. The current treatment of severe cataract is the replacement of the lens by surgery. Oxidative stress may be the cause of senile macular degeneration and cataract, so antioxidants (such as vitamin C) Can be used to treat or prevent these diseases. Studies have proven that vitamin C may slow the age-related macular-34-201249477

Evans, Cochrane Database Syst. Rev·, 2006, (2): CD000254) » In addition, prospective studies have shown that dietary vitamin C reduces the risk of senile cataract in Japanese middle-aged women (Yoshida, et al., Eur. J. Nutr·, 2007, 46:1 1 8- 1 24 ; and Rautiainen, et al·, Am. J. Clin. Nutr., 20 1 0, 1 9 ( 2 ) : 4 8 7-493 ) . Since vitamin C is a recognized antioxidant, on the one hand, the method of treating or preventing a disease or condition using vitamin C captured in a liposome encompasses any disease or condition that can be treated or prevented with an antioxidant. 2. Glutathione Glutathione (GSH) is a tripeptide synthesized from amino acid cysteine, glutamic acid and glycine. Glutathione contains a peptide bond between the amino group of cysteine and the carboxyl group of the glutamate side chain. 1 to 〇 〇 millimolar concentration of glutathione found in bacteria and mammalian cells and acts as a thiol buffer (keeping the cysteine residues of cellular proteins in a reduced state) (US Patent 7,785,900) ). Glutathione exists in two basic forms: antioxidants or "reduced" glutathione tripeptides are generally referred to as "glutathione" or "GSH"; oxidized forms are known as disulfide A sulfur-sulfur bonding compound of glycopeptide (GSSG). Glutathione is a major in vivo autoantibody produced by cells that is directly involved in the neutralization of free radicals with reactive oxygen compounds and in the maintenance of exogenous antioxidants such as vitamins C and E in their reduced (active) form. Glutathione acts as an antioxidant, antitoxin and protectant for red blood cells, and is important for the immune system (US Patent No. RE40849). Glutathione neutralizes free radicals and minimizes damage caused by free radicals. - 201249477 Small, so automatic adjustment of the cells is important. The concentration of reduced glutathione in mammalian cells is associated with a wide variety of pathophysiological states including: cancer, hepatic insufficiency, malignancy, AID S, trauma, burns, sepsis, lung disease , Parkinson's disease, diabetes, Alzheimer's disease, schizophrenia, bloated fibrosis, heart attack and stroke, seizures, sickle cell anemia, bipolar disorder, chronic fatigue syndrome, autism and Immune-related diseases and physiological conditions (eg Kidd, Alternative Medicine Review, 1 997, 2(3): 156-176)0 Direct supplementation of glutathione due to the inability of oral glutathione to be properly absorbed by the gastrointestinal tract It is difficult (WitsChi, et al. (1992), European Journal of Clinical Pharmacology, 43 (6): 667-669). US Patent No. 7,44,096 describes a delivery system comprising a gluten that allows covalent attachment by PEG. The glycopeptide and the phospholipid are covalently bound and then inserted into the liposome carrier. Furthermore, studies of the distribution of glutathione of liposomes in the administration of the lungs indicate that the glutathione is confined to the lung (Romet et al., Int. J. Pharma., 63 (3):227 -235). LipoceuticalTM Glutathione is in the form of an oral solution, and liposomally encapsulated glutathione is currently commercially available. Each teaspoon (5 mL) contains 422.7 mg of glutathione (according to the ingredient table). There is no Recommended Dietary Allowance (RDA) for cumin, but there are no known harmful side effects of the supplement (URL vitaminstuff.com/glutathione.html). In the implementation system, an effective amount of glutathione captured in the liposome is administered. Treatment or Pre-36-201249477 The precise dose required for prevention can be determined by observation of the dose of the test and clinical response. Generally, an effective amount administered will range from about 0.1 mg/kg body weight to about 50 mg/kg body weight. In other embodiments, the administration is from about 0.5 mg/kg to about 25 mg/kg per day. In a further implementation system, approximately 50 to 1,000, 50 to 2,000, 50 to 3,000, 50 to 4,000, 50 to 5,000, and 50 to 10,000 mg per ounce are administered. Conveniently, about 50 to 500 mg per day is administered. a. Method of using glutathione captured in liposome Cancer On the one hand, glutathione captured in a liposome is administered to prevent, treat or ameliorate the signs of cancer. Many cancers develop due to damage to cellular DNA, which results in uncontrolled and autonomous growth and proliferation of damaged cells. One theory is that exposure to free radicals often causes damage to cellular DNA. As an antioxidant, glutathione reacts with free radicals to prevent free radicals from contacting DN A, thereby preventing cancer damage. Preliminary results from recent studies indicate that glutathione changes the concentration of reactive oxygen species in the growth of isolated cells in the laboratory, which may reduce cancer development (Park, 0ncolo gy Reports, 20 09, 3 8 5 -3 9 1 ) . Animal studies in 1 99 3 showed that glutathione inhibited the development of oral cancer. Oral supplementation of glutathione to animals after exposure to DMBA demonstrated significantly fewer tumors than untreated controls (Yance, et al, Herbal Medicine, Healing and Cancer, Keats Publishing, 1999). Further studies have shown that women receiving chemotherapy (cisplatin) and ovarian-breasted glutathione-37-201249477 cancer women not only have fewer side effects of chemotherapy and can tolerate more than one treatment cycle, but also Good overall survival rate (Yance et al.). The glutathione administration with conventional radiotherapy and chemotherapy for the treatment of cancer has been shown to improve the forecast. In the case of colorectal cancer, glutathione administration with oxalic acid platinum/5-fluorouracil/methionine tetrahydrofolate induction reduced neurological deficits compared to administration alone (Milla et al., 2009, Anticancer) Drugs, 20 ( 5 ) : 396-402 ). In the implementation system, glutathione captured in the liposome is co-administered before, after or simultaneously with the chemotherapeutic agent to reduce, prevent or reduce the toxic effects of the chemotherapeutic agent. The glutathione can be co-administered with a chemotherapeutic agent, including but not limited to: vinca alkaloids such as vincristine and vinblastine, and Paclitaxel and its derivatives such as taxotere, anthracyclines, and platinum-based drugs such as cisplatin (U.S. Patent No. 5,618,823, incorporated herein by reference).

AIDS acquired immunodeficiency syndrome (Aids) refers to many infections and signs caused by damage to the immune system caused by exposure to the human immunodeficiency virus (HIV) retrovirus. As far as the disease progresses, the affected patients are increasingly at risk of opportunistic infections and tumor growth. In addition, HIV-infected patients have higher oxidative stress, endothelial cell dysfunction, and cardiovascular disease incidence than uninfected patients (Kline, et al., Am. J. Physiol. Heart Circ. Physiol., 2008, 294 (6) ) : H 2 7 9 2 . H 2 8 0 4 ). The study of the HIV-1 rat model with -38-201249477 showed that the glutathione concentration was restored by the administration of glutathione precursor to allow the effect of interfering with HIV-1 protein on peroxide and NO and vasodilation. Inversion (Kline et al.). Administration of glutathione may also treat AIDS by promoting the immune system and preventing opportunistic infections. Treatment of isolated blood cells from HIV-infected patients with glutathione precursors leads to improved control of intracellular M. tuberculosis infection (Venketaraman, et al., 2006, AIDS Res. Ther., 3:5). Glutathione has also been shown to have antiviral effects in advanced viral replication (〇^^丨, "&1.,1 997,1· Leukoc. Biol·, 62 (1):54-59). Infected with HIV The patient's low glutathione concentration predicts that other indistinguishable HIV-infected patients have a low survival rate (Garaci et al.). In one embodiment, glutathione captured in liposomes is administered for treatment or Prevention of HIV infection or AIDS. In another implementation system, administration of glutathione captured in liposomes to treat or prevent opportunistic infections and burns associated with HIV and AIDS is a skin injury. Caused by exposure to heat, electricity, chemicals 'light, radiation, friction or cold. Burns can cause shock, infection and respiratory distress. Habitually classify burns as first, second, third, and Fourth, fifth or sixth degree. The first degree of burn is usually limited to the surface damage of the epidermis. The second degree of burn contains the superficial layer of the dermis (the papillary layer) and may also contain the deep layer of the epidermis. Layer). The second degree of burn can also be packaged. Nerve damage. Third degree burns include epidermal loss and subcutaneous tissue damage of 39 - 201249477. Fourth degree burns include dermis destruction and muscle damage. Fifth degree burns include destruction of epidermis, dermis and subcutaneous tissue. Sixth degree burn contains epidermis, dermis Subcutaneous tissue and muscle destruction. These burns may also include bone coking. The new classification classifies burns as thickness (shallow and local) and depth (shallow and deep). In addition to the immune system, glutathione Its use has proven effective in saving stagnant areas (one of the targets of burn treatment) (Zor, et al., 2005, Burns, 3 1 (8): 972-976). In a system of implementation, arrested The glutathione is administered in liposome to treat burns. 3. Bran Glutathione α-dithiooctanoic acid (ALA) is an antioxidant found in humans. It is different from other water-soluble or fat-soluble ones. The oxidant, ALA, is water soluble and fat soluble. Evidence also suggests that ALA has the ability to regenerate other antioxidants (Jones et al., Free Radic Biol Med., 2002, 3 3 (1): 8 3 -93 ) » ALA of The C6 carbon atom is chiral and the molecule exists as R mirror image isomer and S mirror image isomer. R-(+)-dithiooctanoic acid (R-LA) is required for several granulocyte enzyme complexes. A cofactor that catalyzes and produces a critical reaction related to the catabolism (decomposition) of a- and keto acids with amino acids (Bustamante, et al., Free Radic Biol Med. 1998, 24 ( 6) :1023-1039) »R/S Racemic mixture (R/S-LA) and R-LA are commonly available in the United States as nutritional supplements, including capsules, lozenges and water. Liquid form. In addition, liposomal A L A protects -40- 201249477 skin cream is commercially available (Reviva Labs). Although ALA is present in many foods, the consumption of ALA from food has not been found to result in an increase in free ALA in human blood prizes or cells (Hermann et al., Eur J Pharm Sci. 1996, 4: 167-174). ). Conversely, a high oral dose of free ALA (50 mg or more) resulted in a significant but transient increase in free ALA in plasma and cells (lpi.oregonstate.edu/infocenter/othernuts/la/). Human pharmacokinetic studies have found that approximately 30 to 40% of R-LA oral doses are absorbed (Hermann et al.). Alpha-dithiooctanoic acid is available in dosage forms from 30 to 100 mg tablets. There is no RDA for ALA, but there are no known harmful side effects of nutritional supplements when ingesting the indicated dose. High doses may cause stomach upset, nausea, diarrhea, and flatulence (see nutritional-supplements-health-guide.com/alpha-lipoic-acid-side-effects.html) 〇In the implementation system, the effective amount of ALA administration in the liposome was taken. In one embodiment, the liposome composition comprises a R mirror image isomer of ALA. In another embodiment, the liposome composition comprises a racemate mixture of ALA. The precise dose required for treatment or prevention can be determined by observation of the dose of the test dose and clinical observation. In the implementation system, about 10 to 10,000 mg is administered daily. In a further implementation system, each 曰 is approximately 1 〇 to 50, 1 0 to 1 0 0, 1 0 to 50,000, 1 0 to 1,000 ' 20 3\ 100, 20 to 500, 20 to 100, 50 To 100, 50 to 2 00, 5 0 to IJ 3 00, 50 to 400, 50 to 6 00, 50 to 8 00, 50 to 900, 50 to 1, 〇〇〇, 50 to 2,000, 50 to! 1 3,000, 50 to 4,000, 50 to 5,000, and 50 to 10,000 mg. Conveniently, -41 - 201249477 Each dose is administered at approximately 50 to 500 mg. For general antioxidant benefits, 20 to 50 mg per day is administered in one implementation system. It will be appreciated that the dose can be administered in divided doses two or more times per day. In one non-limiting embodiment, the liposomes contain about 2.5 to 10 wt% ALA (Na ALA or Na R-ALA). In other embodiments, the liposomes contain at least about 2.5 to 5 wt% ALA. Preferably, it may have less than about 7.5 wt% to prevent or reduce phase separation. As described in Example 9, we found that the liposomes were highly stable and retained 90% of the captured ALA in the liposomes after 50 days of storage at room temperature or elevated temperature (40 °C). In a non-limiting embodiment, the ALA captured in the liposome is stable for at least about 1 to 3 months. In a convenient embodiment, the ALA captured in the liposome is stable for at least about 3 months. a. Method of using ALA captured in liposomes In the implementation system, ALA can be used to treat or prevent oxidative stress or damage (Toklu et al., J. Spinal. Cord Med., 2010, 3 3(4) :40 1 -409 ), diabetes, liver disease, inflammation (Odabasoglu, et al., Br. J. Nutr., 2010, Nov. 15:1-12), neurodegenerative diseases such as Alzheimer's disease (Zara, et Al., Exp. Gerontol., 2010, Nov. 8) 'Cardiovascular disease, peripheral nerve injury (Ranieri, et al., J. Brachial. Plex. Peripher. Nerve Inj., 2010, 5:15), schizophrenia Disease (Seybolt, Med. Hypotheses, 2010, 7 5 (6): 572-575), Moonberry fat (Carbonelli, et al., Curr. Pharm., Des., 2010, 16 ( 7) -42- 201249477 : 840-846), cancer (Schwartz, Oncol., Rep., 2010, 23(5): 1 407- 1 4 1 6 ) and hypertension (Kizhakekuttu, et al., Cardiovasc., Ther·, 2010, 28 ( 4) :e20-e32 ). Diabetes has shown that high doses of ALA can improve glucose utilization in patients with type 2 diabetes. In a clinical trial of 13 patients with type 2 diabetes, a single intravenous infusion of 1 000 mg of racemic ALA resulted in 50% insulin-stimulated glucose utilization (insulin sensitivity) compared to placebo infusion ( Jacob, et al., Arzneimittelforschung. 1995, 45 (8): 872-874) » In a further placebo-controlled study of 72 patients with type 2 diabetes, oral doses of 600 mg/曰, 1 after 4 weeks of treatment Racemic ALA at 200 mg/day or 1 800 mg/day improved insulin sensitivity by 25% (Jacob et al., Biofactors, 1 999, 10 (2-3): 169-1 74). Studies have shown no significant difference between the three doses of ALA, indicating that 600 mg/day may be the maximum effective dose (Ziegler et al., Treat Endocrinol·, 2004, 3(3): 173-189). It has been further demonstrated that ALA can reduce blood glucose concentrations in diabetic patients (Melhem et al., J Am Soc Nephrol., 2002, 13: 108-1 16). The ability of ALA to remove free radicals helps patients with diabetes-induced neurological damage (peripheral neuropathy) reduce pain, fever, itching, stinging and numbness (Androne et al., In Vivo, 2000, 1 4 ( 2 ) : 3 2 7 -3 3 0 ) » ALA in Europe has been used to treat peripheral neuropathy for many years. In another embodiment, liposomal ALA is used to treat vascular disease associated with or associated with diabetes. The vascular lining (called the endothelium) plays an important role in the prevention of vascular disease. Endothelial function in diabetic patients is often damaged and is a high risk group for vascular disease (Schalkwijk et al., Clin Sci (Lond)., 2005, 109(2): 143-159). Racemic LA arterial infusion has been shown to improve endothelium-dependent vasodilation in diabetic patients, but has not improved hygiene (Heitzer et al., Free Radic Biol Med., 2001, 31 (1): 53-61) ° ALA It is also used to treat diabetes-related conditions - autonomic neuropathy, which supplies the nerves of the heart. One study found improvement in 73 patients with autonomic neuropathy compared with placebo in the oral administration of 800 mg of alpha-dithiocyanate (umm.edu/altmed/articles/alpha-lipoic-000285.htm). In one embodiment, at least about 600 to 800 mg/day of ALA captured in a liposome is administered to a patient suffering from diabetes, diabetic neuropathy, and/or diabetic vasospasm. Liver disease ALA combined with silymarin and selenium have been used to treat hepatitis C (Berkson, Med Klin, 1 999, 94 (Suppl. 3): 84-8 9 ). In this study, antioxidants were used to treat three patients with chronic hepatitis C infection with secondary cirrhosis, portal hypertension, and esophageal varices. In one embodiment, ALA, which is captured in a liposome, is administered to treat liver disease and/or hepatitis. Stroke -44- 201249477 Because α-dithiooctanoic acid can easily enter the brain, it has a protective effect on brain and nerve tissue. ALA is being investigated as a potential treatment for stroke and other brain diseases that involve free radical damage. Animals treated with alpha-dithiooctanoate (for example) are less likely to suffer from brain damage and have a fourfold greater survival rate after stroke than animals that do not receive this supplement (Panigrahi et al., Brain Res., 1996, 717 ( 1-2) : 184-188). In one embodiment, ALA, which is captured in a liposome, is administered to treat a stroke.

HIV/AIDS ALA supplementation also has a positive impact on patients with HIV and AIDS by restoring total glutathione levels and making lymphocyte functional responsiveness to T cell mitogens (Jariwalla, et al. , 2008, J. Altern. Complement Med., 14 (2): 139-146). In one embodiment, ALA administered in a liposome is administered to treat HIV and AIDS. » B. Agents Agents suitable for use in cryoplasty to capture liposomes include natural and synthetic compounds having the following therapeutic agents: Arthritis agent, antiarrhythmic agent, antibacterial agent, anti-parasympathetic drug, anticoagulant, antidiuretic, antidote, antiepileptic, antifungal, anti-inflammatory, anti-metabolite, anti-migraine agent , anti-tumor agents, anti-parasitic agents, antipyretics' anti-epileptic agents, anti-serum, tinctures, analgesics, anesthetics, beta-blockers, biological effect modifiers, bone metabolism regulators, cardiovascular drugs, Diuretic, yeast-45- 201249477 素, fertility drug, growth promoter, hemostatic agent, hormone, hormone inhibitor, hypercalcemia mitigator, hypocalcemia mitigator, hypoglycemia mitigator, hyperglycemia mitigator, immunosuppression Agents, immune enhancers, muscle relaxants, neurotransmitters, parasympathomimetics, sympathomimetic plasma diluents, plasma prolongers, psychotherapeutics, thrombolytics, and blood vessels Zhang agent. In one embodiment, the agent is a cytotoxic drug or agent, an anthracycline antibiotic is selected from the group consisting of doxorubicin, doxorubicin, epirubicin, and idarubicin; and platinum compounds include, but are not limited to, Cisplatin, carboplatin, ormaplatin, oxaliplatin, zeniplatin, enloplatin, lobaplatin, spiroplatin ), ((-)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutyldicarboxylic acid)platinum) (DWA2114R), (SP-4-3(R)-l,l - (2-)-(2-methyl-1,4·butylenediamine...Ν,Ν·)platinum) (CI-973), nedaplatin (25 4-S) And (diacetate-ammonium complex-dichloro-cyclohexylamine-uranium (IV)): Type 1 topology isomerase inhibitor is selected from, but not limited to, topotecan, Liticon (Bu 1|1〇16〇311), (7-(4-methylpiperazine-extension methyl)-10,11-Exoethylenedioxy-20(S)-camptothecin), 7 -(2-(N-isopropylamino)ethyl)-(20S) · camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin; long Flower bioassay is selected from (but not limited to) Vincristine, vinblastine, vinleurosi ne, vinrodisine, vinorelbine, vinca vindesine Angiogenesis inhibitors include, but are not limited to, vascular occlusion - 46-201249477 angiostatin, endostatin, and tumor necrosis factor (TNF-α); and DNA nucleic acids and RNA nucleic acids. The single dosage form provides a single dosage form of a nutritional supplement or composition that is captured in the liposome in a suitable container in one embodiment. Conveniently, the container is a suitably sized pouch or package containing the single dosage form. The container may be formed from paper (coated or uncoated), foil, polymer, metal, plastic, puncture resistant nylon film or any combination thereof. In the practice system the container is formed from a foil laminate, a heat seal laminate or a ribbon package. The container may be formed from one or more layers of suitable materials. The container may also be formed from a different layer of suitable material. In one embodiment, the container is formed from one or more layers of suitable materials. For example, the inner layer in contact with the supplement or composition may be formed from an inert material that does not react with the ingredients of the supplement or composition. The inner layer may also be formed from a material that is impermeable to water, gas, and/or light. The outer layer may also be formed from a material suitable for the logo. In one embodiment, the granulator is formed from multiple layers. In this embodiment, the inner layer may be formed of a material that provides a vapor barrier, prevents evaporation of the composition, and/or prevents degradation by ultraviolet light and other light sources. In one embodiment, the inner layer is a material such as aluminum. In another embodiment, the container is formed from one or more layers of thermoplastic polymer. Suitable thermoplastic polymers include, but are not limited to, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polytetrafluoroethylene, acrylonitrile butadiene styrene, and polyamine or a combination thereof. Where the container is formed from a sheet of material, the sheet of material is conveniently folded - and sealed along at least the side to leave an opening to fill the single dose. Where the barn is formed from two or more sheets of material, the two sides are sealed by any suitable means to leave an opening to fill the single dose. In this manner the container has internal cavities for filling a single dose. Exemplary containers are shown in Figures 2A through 2A. It is seen in Figure 2A that the container 10 can be formed by sealing the edges 12 leaving the inner 14 unsealed face-to-face two sheets. It is seen in Figure 2B that one side is initially unsealed to form an inflatable region 16 for containing a product such as a liposome composition. This side can be further sealed after filling the container. The liposome composition of the appropriate agent fl is dispensed into the interior of the container. Once the liposome composition is dispensed into the container, the remaining edges are sealed. The container can be sealed under an inert gas such as N2. Sealing the container under inert gas improves the stability of certain captured agents, such as vitamin C. The sides of the container may be sealed by any suitable means including heat sealing or the use of a suitable adhesive. The sides of the container may be used in a quantity sufficient to allow at least a portion of the material to at least partially melt to form a heat and heat seal of the container. In this way, the sides of the container are stacked together. In other implementations, the container may be heat sealed with sonic welding. In another embodiment, the container can be sealed with a suitable adhesive. Suitable adhesives include, but are not limited to, acrylic polymers and copolymers, natural latex rubbers, and vinyl acetate. The container can be further sealed with a zipper. The container may further include a peeling portion to allow easy opening of the container after sealing. VI. EXAMPLES -48- 201249477 Essentially the following examples are illustrative and are in no way intended to be limiting. Example 1 Preparation of sodium ascorbate captured by liposome 97 g of Phospholipon 50IP (a phospholipid choline lipid for forming a small capsule commercially available from Lipoid) was dissolved in 56.3 g of 200 proof ethanol at room temperature. ). Care is taken to ensure that all lipids are completely dissolved without undissolved lipid residues. 109.5 g of sodium ascorbate and 26.4 mg of EDTA were individually dissolved in 251 g of reverse osmosis water at room temperature under normal agitation. The pH of the sodium ascorbate-EDTA solution was adjusted to 6.3. The sodium ascorbate-EDTA solution was filtered through a 0.2 μηι filter to remove dust particles and other contaminants. The lipid solution was slowly infused into the vitamin solution with a stirrer under a continuous vortex. After all of the lipid solution was added, the lipid hydration was continued for about 15 minutes with frequent mixing. At this point, the liposomes are formed in a smooth, translucent fluid. 2.2 g of Sanxian gum (commercially available from Sigma Aldrich®) and 1.38 g of TweenTM 80 (commercially available from Sigma-Aldrich®) were added to thicken the solution into a gel. The liposome solution formed is a color that is transparent like honey. Example 2

Comparison of Empirical Liposomes and LivOn Liposomes Several batches of Lypo-SphericTM Vitamin C liposomes are commercially available from LivOn Laboratories. According to the product ingredient list, each 5.7 mL packet of L y ρ 〇- S pheric τ MV itamin C 1 i ρ 〇s 〇 Mes contains 1 0 0 0 mg of -49- 201249477 sodium ascorbate and 1.0 g of essential phospholipids. The Lyp〇-SphericTM Vitamin C liposomal is available in a range of light orange to brown. The second product, Liposomal Vitamin C liposomes, is commercially available from Empirical Laboratories. According to the product ingredient list, each 4 mL portion of Empirical Liposomal Vitamin C liposomes contains 1 000 mg of sodium ascorbate and 400 mg of phospholipid choline. Comparing the sodium ascorbate content, particle size, dry matter content and lipid content of LivOn and Empirical liposomes formulations (estimated), the results are shown in Table 2» Compared with LivOn liposomes, Empirical liposomes appear thin. The Empirical 1 i p o s o m e s is divided into two layers when left on the bench for a relatively short period of time (<1 hours). At the time of centrifugation of the separate Empirical liposomes, the lower layer accounts for approximately 70 to 80% of the total volume. » The second layer of the empirical liposomes is brown and relatively transparent. The lower layer is more like water and the upper layer is more gel-like. By hand shaking the mixture, the Empirical product becomes cloudy and more viscous. The sodium ascorbate content in the liposome was determined by HPLC. The sodium ascorbate was first separated by centrifugal filtration to determine the sodium ascorbate conjugation. The 200 pg product was weighed and diluted with 10 mL of 0.5 M NaCl solution. The mixture is vortexed to allow the liposomes to be completely dispersed in the buffer. This solution was further diluted 1 time with 80/20% ethanol/water to obtain a clear solution, and the total ascorbate concentration was analyzed by HPLC. Load a small amount of approximately 〇5 to 1 mL of the solution into a centrifugal concentrator (Nanosep® 10K OMEGA p/n OD010C34 or Millipore's Biomax UFV5BTK25 30K commercially available from Pall Corp.) and centrifuge 1 to 2 small-50- The amount of free ascorbate was determined at 201249477. The filter has very fine pores and allows only the aqueous solution containing free (unencapsulated) sodium ascorbate to pass through while retaining the liposomes. The filtrate was then diluted 10 times with 80/20% ethanol/water. The concentration of sodium ascorbate in the filtrate was measured by HPLC and the total sodium ascorbate concentration was compared to determine the encapsulation efficiency. An estimate of the amount of product is dried 2 to 3 inches in a freeze dryer to assess the dry content of the liposomes. The weight change after vacuum drying was measured. The particle size was measured by dynamic light scattering (NICOMP 3 70) after dilution with saline. The solution was vortexed to ensure complete dispersion of the liposomes. Three measurements were made for E m p i r i c a 1 1 i ρ 〇 s 〇 m e s to obtain an average particle size of 6 2 0 to 760 nm and a standard deviation of 460 to 615 nm. The particle size of two individual batches of LivOn liposomes was measured. Compared to the L1208 batch (old), the L0809 batch (new) has an average particle size of approximately 1200 ηιη and a wider distribution (SD 760 nm). The L1208 batch has an average particle size of approximately 800 nm and a standard deviation (SD) of 45 0 nm. In contrast, the particle size of liposomes prepared by the cold working described in the present invention is typically in the range of from 300 to 500 run. In individual experiments, the particle size of 17 containers from 5 batches of LivOn liposomes was measured. The average particle size is from less than 850 nm to more than 1200 nm with an average particle size of 1030 ± 100 nm. There was no statistical difference in particle size between the batches tested. The peak width of the particle size averaged approximately 740 ± 60 nm with no statistical difference between batches. A small amount of product was diluted with MilliQ water to measure pH 値. The mixture is vortexed to completely disperse the liposomes. Measured using a standard laboratory pH meter -51 - 201249477 pH値. Observation of Empirical liposomes and LivOn liposomes under microscopic observation. The Empirical liposomes have many very large oil droplets. Since the oil droplets are essentially transparent to the laser beam of the particle classifier, the particle size measured in Table 2 does not account for the effect of the oil droplets. The list of ingredients of the empirical liposomes includes olive oil, vitamin E and flavorings. Any of these ingredients may account for large oil droplets seen in the product. Furthermore, if low grade lipids are used in the manufacture of Empirical liposomes, the oil may be derived from the lipid material. The Empirical liposomes solution diluted under microscopic observation appeared to be less uniform than the LivOn liposomes solution. Table 2: Empirical Laboratory Liposomes and LivOn

Comparison of Liposomes

Empirical Laboratories Lipo C (dose 4.58g or 4mL) LivOn Laboratories Lypo-SphericTM Vitamin C (6.5g or 5.7mL) Ascorbic _(w/w%) 19.9 ±0.6 17.1 ±0.3 Sodium Ascorbate _) 09.1 ±0.02 1.11 ±0.04 Ascorbic acid _) 0.81 ±0.02 0.99 ± 0.04 Sodium ascorbate 7.4 ± 2.4 28 ± 1.4 Encapsulation (%) Particle size (nm, average ± SD) 660 ± 510 1180 ± 760 * Dry content (w/ w%) 40 ± 0.5 41 ±0.5 lipid content (estimated, w/w%) 18.2 ± 0.2 19.5 ±0.3 pH 7.0 5.5 The old batch tested was of 800 nm ± 450 nm -52-201249477 Example 3 Cold Process Liposomes and LivOn Laboratories Liposomes The cold processed liposomes were essentially prepared as described in Example 1. Lypo-SphericTM Vitamin C liposomes is commercially available from LivOn Laboratories. The various properties of the liposome formulation were determined and the results are shown in Table 3. The weight percentage of ascorbic acid in the liposome formulation was determined by HPLC. The ethanol content was determined by gas chromatography. The average particle size was determined by dynamic light scattering. Ascorbate was encapsulated by filtration and HPLC as described in Example 2. Table 3: Ratio of Cold Process Liposomes to LivOn Liposomes _ Cold Processed Liposomes LivOn Laboratories Lypo-SphericTM Vitamin C Vitamin C (ascorbic acid)% 19.6% 16.5% Pity (Wt%) 18.8% 18.7% Ethanol 10-11% 9-10% average particle size (nm) 300-450 800-1200 particle size distribution SD (nm) 150-200 600-900 sodium ascorbate cystic (%) 35 35 pH 6.3 6.0-6.5 at 50 95% after the incubation at °C (13 days) 94% (13 days) Change in Cwt0/〇 (% of T=0) 94% (19 days) 90% (19 days) Yellowish-like honey Gel Brown Yellow Gel Example 4 - 53 - 201249477 Liposomal Stability at Room Temperature and High Temperature The storage stability of liposomes prepared by cold working was investigated. The liposomes were prepared essentially as described in Example 1, but using Alc〇lec pc 50 lipid (GMO phospholipid choline lipid commercially available from American Lecithin Company) instead of non-GMO lipids. Capsules were filled with 2.0 mL plastic bottles with liposomes and sealed with a screw cap under N2 (each cap has a rubber dome for complete sealing). Store the plastic bottle at room temperature (25 ° C) or high temperature (50 ° C). At 4, 5, 7 (two measurements), 8 and 38 days, the liposomes stored at room temperature were measured by HPLC. Ascorbic acid content (wt%). The ascorbic acid content (wt%) of the liposomes stored at high temperature was measured by HPLC at 0, 5, 6 (two measurements), 11, 20 and 42 Å. The results are shown in Table 4. Table 4: Stability of liposomes stored at room temperature and high temperature Stored at 25 ° C Stored at 50 ° C 曰 ascorbic acid (wt % ) 曰 ascorbic acid (wt % ) TO% 4 18.64 0 18.64 100.0 5 18.28 5 18.1 97.1 7 18.7 6 19.19 103.0 7 · 18.8 6 17.43 93.5 8 18.48 11 18.3 98.2 3 8 18.6 20 17.85 95.8 42 15.06 80.8 Example 5 Comparison of liposome stability stored at 50 ° C After storage at 50 ° C Cold-processed liposomes (cold process -54 - 201249477 liposomes) and LivOn liposomes stability. The cold processed liposomes were essentially prepared as described in Example 1. Lyp〇-SphericTM Vitamin C liposomes is commercially available from LivOn Laboratories. The cold-processed liposomes were purged with N2 and stored in a 2.0 mL plastic bottle. LivOn liposomes were stored in the original packaging or removed from the original packaging, washed with N2, and stored in a 2.0 mL plastic bottle. The weight percentage of ascorbic acid was determined by HPLC. The liposomes were then stored at 50 °C. The liposome composition was removed after the 4th, 13th or 19th day, and the weight percentage of ascorbic acid was determined by HPLC. The results are shown in Table 5. Figure 4 is a comparison of liposomes prepared by cold working and Lypo-f commercially available from LivOn Laboratories.

SphericTM Vitamin C liposomes A map of stability within 19 days. Table 5: Cold Process Liposomes and LivOn Liposomes in 50% stability cold-processed liposomes (in plastic bottles, purged with N2) LivOn liposomes (in plastic bottles, purged with N2) LivOn lipids Body (in the original packaging) 曰 ascorbic acid (wt%) TO%% Ascorbic acid (wt%) TO% Ascorbic acid (wt%) TO%% 0 19.6 100.0 16.7 100.0 16.7 100.0 4 18.1 92.3 16.5 98.8 n/dn/d 13 19.19 97.9 15.7 94.0 16.6 99.3 19 17.43 88.9 15.0 89.8 16.6 99.5 A large (14 kg) amount of sodium ascorbate trapped in liposomes was produced in order to fully evaluate the stability of liposomal ascorbate prepared by cold working. The sodium ascorbate content was determined by the weight of the ingredients to be 22.1 - 55 - 201249477 wt%. The manufacturing method was similar to that described in Example 1, except that a large container (5 gallon drum) was used to dissolve the lipid and vitamin C. The industrial grade homogenizer/mixer was used to mix the liposomes. The final product looked similar to the final product obtained in Example 1. Each LivOn Labs patch was filled with 5.5 g of the final liposomal ascorbate in exactly the same manner as the commercial Lypo-SphericTM Vitamin C liposomes package. Ascorbate stability was monitored at 25 ° C and 4 (TC). HPLC analysis results are shown in Figure 6. Example 6 Preparation of reduced glutathione captured in liposomes 48 g of 200 proof ethanol at room temperature Dissolve 80 g of Ale olec PC 50. Take care to ensure that all lipids are completely dissolved and that there are no undissolved lipid residues. Let 80 g of reduced form be treated with 0.9% NaCM, 50 mM Ρ04 (ρΗ 3.9) at room temperature and under normal agitation. Dissolve glutathione (GSH). Add 120 mg of EDTA to glutathione solution and dissolve completely. Slowly inject a solution of ethanol lipid into glutathione solution with continuous mixing under high speed mixer to make The glutathione is captured in the liposome. After adding the entire lipid solution, the lipid hydration is continued for about 30 to 60 minutes with frequent mixing. At this time, the liposome is formed in a smooth yellow turbid fluid. The particle size is measured by a dynamic light scattering instrument (ZetaPALS (Broughhaven). The particle size is usually in the range of 200 to 600 nm. 1.6 g of Sanxian gum (commercially available from Sigma-Aldrich®) is incorporated into the mixture. In the liposome, followed by the incorporation of 2.0 g of Twee n 80 (from -56 to 201249477

The Sigma-Aldrich® is commercially available) to thicken the liposome into a gel. It is a thick transparent gel like honey. The final product has a 20.0 glutathione concentration. Example 7 HPLC determination of reduced glutathione in liposomes Reduced glutenase in liposome glutathione formulations prepared in 6 was determined by HPLC analysis developed according to the published method (Raggi et al. , 1 997, Chromatographia, vol. 46, 1 The measured glutathione concentration was 18.4 ± 0.3 wt%, very close to wt%, indicating the stability of the starting glutathione starting material during the formulation process. Example 8 Preparation of α-dithiooctanoic acid captured in liposomes AlcolecTM PC 50 lipid was dissolved in 12 g of 200 proof ethanol at room temperature. Care was taken to ensure that all lipids were free of undissolved lipid residues. And generally agitate the next 0.3 ° / EDTA of water (pH 7.5) to dissolve 5.0, 7.5 or 10.0 sodium lipoate (Na ALA) or 5.0, 7, 5 or 10 g of the ruler - (Na R-ALA). The continuously mixed lipid solution was slowly injected into the Na R-ALA solution by a high-speed mixer to produce Na R-ALA in the trap. After adding the entire lipid solution, the lipid hydration was allowed to continue for about 30 to 60 minutes. The final product wt% of the determined example glycine concentration 7-22) » Theory 値 20 92% 20 g of almost totally dissolved and not containing α g is used - when the lower two salt ALA ethanol, collected frequently mixed lipid, liposomes -57-201249477 formed in a smooth yellow turbid fluid. 0.4 g of Sanxian gum was incorporated into the liposome under mixing, followed by the incorporation of 0.3 g of Tween 80 to thicken the liposome into a gel. The final product was a thick, turbid yellow gel. The final product has a theoretical concentration of Na ALA or R-ALA of 5, 7.5 and 10.0 wt%. Example 9 HPLC determination of liposome potency and stability of dilipoic acid Na-ALA and R-ALA liposome stability as described in Example 8 were investigated. For stability monitoring, the liposome formulation is used to fill the glass bottle without leaving a headspace to minimize contact air and seal with a screw cap. Store samples at room temperature or 40 °C. It was found that after several days, several phases were separated in the 10% Na-ALA and 10% Na R-ALA formulations, and a clear solution was precipitated in the lower half of the vessel. 7.5% Na ALA and Na-R-ALA also have significant but minor phase separations, while 5 wt% formulations remain homogeneous for at least 8 weeks by using the GeroNova Research Inc website ( geronova.com/sites/default/files/LA_HPLC_Mod_USP .pdf) The method developed by the internal method of "Analysis of Lipoic Acid Salts for Quality Assurance" measures the dithiooctanoic acid efficiency of a formulation containing 5 wt ° / 〇 Na-ALA and 5 wt% R-ALA. The results of the analysis are shown in Table 6 below. The results show that the ALA obtained from the liposome obtained by cold working is very stable, indicating that the ALA or R-ALA efficacy is reduced less than 50 days after incubation at room temperature or 40 °C. 10%. -58- 201249477 Table 6: Stability formula of Na-ALA and R-ALA liposomes stored at room temperature and 40 ° C. ALA/R-ALA potency ratio stored at room temperature (wt%) stored at 40 ° C ( Wt%) 5 wt% Na-ALA liposome 5.19% 2nd day 5_38% 2nd 4.87 % 50th day 5.27 % 50th day 50th / 2nd day 94.0% 98.0% 5 wt% Na-R-ALA Liposomes 5.15% Day 2 5.60% Day 2 4.74 % Day 50 5.14 % Day 50 Day 50 / Day 2 92.0% 91.7% In individual studies, three were prepared as described in Example 8. A Na-ALA liposome formulation containing 2.5, 5.0 or 7.5 wt% Na-ALA. For stability monitoring, the liposomes were filled with the liposome without leaving a headspace and sealed with a screw cap. Similarly, phase separation occurred in the 7.5 wt% ALA and 7.5 wt% R-ALA formulations. The formulation of 5.0 wt% and 2 · 5 wt% remained homogeneous throughout the study. The sample was stored at room temperature or 40 ° C and the ALA concentration was determined by HPLC. The results are shown in Table 7. The results indicate that ALA obtained by cold working and captured in liposomes is very stable for at least three months. The results further indicate that in all cases (even if the formula is stored at 40 °C) the ALA effect is reduced by less than 1% from the end of the first month to the end of the third month. Due to the phase separation of the formulation resulting in non-uniform sampling, the HPLC measurements of the two 7.5% formulations were less accurate due to the inefficiency of the HPLC method in preparing these formulations without T = 0. -59- 201249477 Table 7: Na-ALA concentration in liposome after letter 1 stability formulation # Theory Na-ALA (wt%) Na-ALA at room temperature (wt%) Na-ALA at 40 °C , by HPLC (wt%) 1 7.5 6.52 % 1 month 8.26 % 1 month 5.63 % 3 months 7.78 % 3 months comparison (3 months / 1 month) 86.4% 94.1% 2 5.0 5.25 % 1 month 5.66 % 1 month 4.83 % 3 months 5.Π%3 months comparison (3 months/1 month) 91.3% 92.1% 3 2.5 2.62 % 1 month 2.63 % 1 month 2.42 % 3 months 2.53 % 3 months comparison (3 months / 1 month) 92.4% 96.3% When some of the demonstration aspects and implementation systems are discussed above, some artists will recognize some modifications and replacements. , additions and sub-combinations. Therefore, we intend to understand the scope of the patent application and the scope of the patent application filed in the following, including all modifications and substitutions in the true spirit and scope. All of the aforementioned patents and publications are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A to 1B show the chemical structures of L-ascorbic acid (Figure 1A) and L-ascorbate (Figure 1 B). 2A through 2B show side and top views of an exemplary container. Figure 3 shows the stability ( ♦) of sodium ascorbate trapped in liposomes stored at room temperature for vitamin C (wt%) versus time (days) and stored at 50 °C. The stability of sodium ascorbate captured in liposomes (). Figure 4 shows liposome stability () prepared by the implementation system of the method for the vitamin C (wt%) on the 19th, stored in a 50 ° C bottle, stored in 5 (TC of the vial) Commercially available sodium ascorbate (LivOn Lypo-SphericTM Vitamin C) obtained from liposomes (♦), and commercially available in the original packaging of 5 (TC), captured in liposomes Sodium ascorbate (LivOn Lypo-SphericTM Vitamin C) stability (▲) Figure 5 shows the preparation of the vitamin C (wt%) for the 90th day of the vial stored at 50 °C by the implementation system of the method Liposomal stability (♦), commercially available sodium sulphate (LivOn Lypo-SpheriCTM Vitamin C) stability () stored in a lipohydrate bottle at 50 ° C, stored at 50 ° The commercially available sodium ascorbate (LivOn Lypo-SphericTM Vitamin C) from the liposome is commercially available (▲). Figure 6 shows the storage of sodium ascorbate (wt%) at 25 a pharmaceutical bottle of °C prepared by the implementation system of the method Solitaire stability ( ♦) and liposome stability prepared by the implementation system of the method in a vial stored at 40 ° C. [Main component symbol description] 10 : Container - 61 - 201249477 12 : Side 14 : Internal 16: Rechargeable area - 62

Claims (1)

201249477 VII. Scope of Application: 1. A method of making liposomes having a captured agent comprising dissolving at least about 18 w/w% of the lipids forming the vesicles at about 1 to 1 2 at room temperature. w/w% in an aqueous solvent to form a lipid solution; additionally, an amount of the agent is dissolved in 0.005 to 0.01 w/w% of EDTA at room temperature to form a solution containing the agent; filtering the solution containing the agent; A portion of the lipid solution is injected into the solution containing the drug while mixing: and the resulting solution containing the lipid and the agent is hydrated for at least one hour with frequent mixing. 2. The method of claim 1, wherein the aqueous solvent is an alcohol. 3. The method of claim 2 or 2, further comprising adding more lipid or thickening agent to form a gel after the hydrating step. 4. The method of claim 1, wherein the lipid for forming the sachet contains at least about 45 to 50% phospholipid choline. 5. The method of claim 4, wherein the phospholipid choline is derived from egg or soybean. 6. The method of claim 1, wherein the agent is ascorbic acid or a salt thereof. 7. The method of claim 1, wherein the agent is glutathione. The method of claim 1 wherein the agent is alpha lipoic acid. A liposome composition for administering ascorbic acid or a salt thereof by the method of claim 6 of the patent application. 1 〇. The liposome composition of claim 9 is composed of the following: at least about 18 w/w% of the lipid forming the smear: 0.005 to 0.1 w/w% of EDTA; 1 0 to 1 2 w/w ° / oxime alcohol; liposomes having a selected average particle size of 200 to 500 nm, and at least about 22 w/w% of ascorbic acid or a salt thereof trapped in the liposome. 11. The composition of claim 9 or 10 wherein the lipid formed into a small birthday is at least about 45 to 50% phospholipid choline. 1 2. The composition of claim 11, wherein the phospholipid choline lipid is soybean phospholipid choline. 1 3. The composition of claim 9 or 10, further comprising 0 to 0.6 w/w ° /. Three immortal gum. 1 4. The composition of claim 9 or 10, further comprising 0.4 w/w % Tween 80. The composition of claim 9 or claim 1, wherein the ascorbic acid or a salt thereof is sodium ascorbate. 1 6. The composition of claim 9 or 1 is used for the treatment of scurvy, cardiovascular disease, cerebrovascular disease, cancer, age-related macular-64-201249477 lesions, cataracts, gout, heavy metals Toxic or diabetic. 1 7 · A unit dose product containing a unit dose package and a liposome composition as claimed in claims 9 to 15 of the package. 18. The unit dose product of claim i, wherein the liposome composition is sealed in the package under N2. 19. A unit dose product according to claim 17 or 18 for the treatment of scurvy, cardiovascular disease, cerebrovascular disease, cancer, age-related macular degeneration, cataract, gout, heavy metal toxicity or diabetes. -65-